Polymer 51 (2010) 2286e2295

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Polymer
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Synthesis and characterization of bioactive molecules grafted on poly(3-caprolactone) by click chemistry

Jiraphong Suksiriworapong a, Kittisak Sripha b, *, Varaporn Buraphacheep Junyaprasert a
a b

Department of Pharmacy, Faculty of Pharmacy, Mahidol University, Bangkok 10400, Thailand Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Mahidol University, Bangkok 10400, Thailand

a r t i c l e i n f o
Article history: Received 1 December 2009 Received in revised form 18 March 2010 Accepted 21 March 2010 Available online 27 March 2010 Keywords: Bioactive molecules Poly(3-caprolactone) Click chemistry

a b s t r a c t
A facile and efcient strategy to graft bioactive molecules (nicotinic acid, p-aminobenzoic acid, and phthaloyltryptophan) onto poly(3-caprolactone) (P(3CL)) was achieved by copper-catalyzed Huisgens 1,3-dipolar cycloaddition known as click reaction. P(aCl3CL), with 10, 20, and 30% of a-chloro-3-caprolactone (aCl3CL) units were copolymerized by ring opening polymerization using 3CL and aCl3CL as starting materials in the presence of 1,4-butanediol and Sn(Oct)2. Subsequently, the chloride pendent was converted to azide followed by cycloaddition with terminal alkyne derivatives of the aforementioned bioactive molecules. The complete addition was accomplished at all ratios. The characteristic molecular features of these copolymers were evaluated by FTIR, NMR, and GPC. Thermal analysis data indicated that the grafted compounds led to polymorphic alteration and different pattern of thermal degradation depending on the molecular structure and the size of the grafted compounds. They are the basis for further development of grafted copolymer as drug delivery carriers. Crown Copyright 2010 Published by Elsevier Ltd. All rights reserved.

1. Introduction Over the past decades, research on grafting bioactive molecules onto biodegradable polymer backbones has gained much interest to augment the efcacy of medical and pharmaceutical applications including drug delivery systems, tissue engineering, and medical devices. Various methods have been used to modify biomaterials for drug delivery. In general, drugs or attractive functional groups may be conjugated to polymers [1e6]. But the amount of the conjugated drug is limited since only one drug molecule or group can adhere at one-side of the polymer chain. The other approach is accomplished by substitution of functional groups such as hydroxyl, amino, and carboxylic acid along the polymer backbone. Although this method seems promising, the protic functional groups lead to intra- and intermolecular transesterication resulting in chain degradation and uncontrolled molecular weight [7]. The polyesters poly(lactide), poly(glycolide), and poly(3-caprolactone) are considered polymers with attractive properties, i.e., biodegradable, biocompatible and environmental friendly properties. Poly(3-caprolactone) (P(3CL)) has been synthesized and functionalized to enhance its attractive properties. Several methods have been proposed to graft functional groups along the P(3CL)

* Corresponding author. Tel./fax: 66 2 644 8677. E-mail address: pyksp@mahidol.ac.th (K. Sripha).

backbone; however, they often encountered limitations such as incomplete conversion [8] or multiple step synthesis [9,10]. Recently, it was reported on the possibility of grafting of functional groups along the P(3CL) through Huisgens 1,3-dipolar cycloaddition, known as click chemistry [11]. This reaction has gained much attention due to its feasible, easy, and mild conditions. Riva et al. [12e14] and Lee et al. [15] demonstrated that small functional groups (i.e., benzoate, triethyl ammonium bromide, hexyne, and proline) and macromolecules (such as Poly(ethylene oxide) (PEO)) could be successfully grafted onto the polymer backbone by the click reaction. In addition, the ligands could be easily conjugated along the polymer backbone with various amounts of grafting units. Although a number of attempts have been made to attach either functional groups or drug molecules on polymer backbones, only a few studies are found concerned with the attachment of drugs onto polyester backbones, in particular onto P(3CL) [16,17]. Therefore the pharmacologically bioactive compounds nicotinic acid, p-aminobenzoic acid (PABA) and phthaloyltryptophan [18e25], shown in Fig. 1, were chosen as model compounds for grafting on P(3CL) to evaluate possible interactions between drug molecule and the polymer during the grafting reaction and to compare the physicochemical properties of the resulting copolymers. Contrasting PABA with nicotinic acid, the pendent amino group of PABA in para position is a protic functional group with basic character acting as hydrogen bond donor; however, it exhibits an acidic property according to its pKa (4.65 and 4.8) [26,27]. The

0032-3861/$ e see front matter Crown Copyright 2010 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.polymer.2010.03.034

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Fig. 1. Structure of bioactive model compounds used in the current study. Fig. 2. Esterication of bioactive molecules with 3-butyn-1-ol using coupling reaction (1 but-3-ynyl nicotinate, 2 but-3-ynyl 4-aminobenzoate, and 3 3(2-N-phthalimido-2-(prop-2-ynyl acetayl)ethyl)indole).

nitrogen atom of the nicotinic pyridine ring on the contrary is solely a hydrogen bond acceptor providing a pair of unshared electrons with basic character. These differences in molecular properties may inuence the grafting reaction and affect drug loading and the release characteristic. In addition, the homo- and heteroaromatic rings of these two molecules can display aromatic pestacking interaction with other loaded drugs in nanoparticles made of these copolymers. The third model drug, phthaloyltryptophan, was selected as an example of a large bioactive compound the bulky structure of which may have steric effects in contrast to the former two drugs. To the best of our knowledge, there is so far no report on the engrafting of these three bioactive molecules (nicotinic acid, PABA, and phthaloyltryptophan) on P(3CL) by click reaction. Therefore, the aim of this work was to study the synthesis of novel engrafted P (3CL) with these compounds by Huisgens 1,3-dipolar cycloaddition, and to evaluate its limitation as well as to compare the differing molecular properties of the grafted copolymers including their thermal properties in ratio to their composition. 2. Experimental section 2.1. Materials

by column chromatography on silica gel using a mixture of ethyl acetate (EtOAc), hexane (Hex), and glacial acetic acid (CH3COOH) (4:1:0.01) as mobile phase. The collected fraction was twice extracted with puried water to eliminate residual acetic acid. The organic layer was then dried again over anhydrous magnesium sulfate, ltered, and evaporated under reduced pressure to yield a yellow solid (0.8 g, 98%); m.p. 172.3  C; TLC Rf 0.13 (silica gel, EtOAc:Hex:CH3COOH/4:1:0.01); FTIR (KBr) n (cm1) 3408, 3056, 2928, 1774, 1710, 1391, 1104, 1071, 718; 1H NMR (CDCl3): d 3.80 (m, 2H, CH2CH), 5.35 (m, 1H, CH2CH), 7.02 (s, 1H, indole), 7.17 (m, 1H, indole), 7.27 (d, 1H, indole), 7.62 (s, 1H, indole), 7.65 (m, 2H, phthaloyl), 7.75 (m, 2H, phthaloyl), 8.03 (s, 1H, NH); MS (EI, 70 eV) m/z (rel int) 335 [M H] and 357 [M Na]; Anal. Calcd. for C19H14N2O4: C, 68.26; H, 4.22; N, 8.38; O, 19.14. Found: C, 68.03; H, 4.18; N, 8.29; O, 19.50. 2.3. General procedure for esterication with 3-butyn-1-ol (Fig. 2) The required amounts of DCC and DMAP were dissolved in (CH2Cl2) in a reaction ask. An acidic model compound was added and stirred at room temperature for 30 min 3-Butyn-1-ol was charged through a dried stainless steel syringe under argon atmosphere. The reaction was conducted at room temperature for 24 h. After ltration of the precipitate, the solvent was evaporated under reduced pressure. The crude product was then puried by column chromatography on silica gel. 2.3.1. Synthesis of but-3-ynyl nicotinate (1) Nicotinic acid (5.0 g, 40.6 mmol), 3.4 g (48.8 mmol) of 3-butyn1-ol, 12.6 g (61.0 mmol) of DCC, and 7.4 g (61.0 mmol) of DMAP gave according to 2.3 an off-white solid. Puried compound 1 (5.9 g, 83%) was obtained through column chromatography on silica gel with the mobile phase of Hex, EtOAc, and CH2Cl2 (1:2:2); m.p. 46.43  C TLC with Rf 0.50, FTIR (KBr) n (cm1): 3264, 2926, 2850, 2119, 1718; 1 H NMR (DMSO-d6): d 2.65 (m, 2H, CH2C^CH), 2.86 (t, J 2.61 Hz, 1H, C^CH), 4.35 (t, J 6.48 Hz, 2H, COCH2), 7.57 (m, 1H, pyridine), 8.28 (td, J 7.96 Hz, 1H, pyridine), 8.80 (s, 1H, pyridine), 9.08 (s, 1H, pyridine);

3-Caprolactone (3CL; Aldrich) was dried over CaH2 for 48 h followed by distillation prior to use. PABA (Aldrich), nicotinic acid (Ajax Finechem), and L-tryptophan (Fluka) were used without any purication. 1,4-Butanediol, 3-butyn-1-ol, a-chlorocyclohexanone, N-carbethoxyphthalimide, copper (I) iodide (CuI), 1,8-diazabicyclo [5.4.0]undec-7-ene (DBU), and stannous (II) octanoate (Sn(Oct)2) were obtained from Aldrich Chemicals. N,N-dicyclohexylcarbodiimide (DCC), 4-dimethylaminopyridine (DMAP), and mchloroperoxybenzoic acid (mCPBA, 70%) were purchased from Fluka. Sodium azide was purchased from Asia Pacic Specialty Chemicals Limited. Dichloromethane (CH2Cl2), dimethylformamide (DMF), and tetrahydrofuran (THF) were dried over molecular sieve 4 A overnight. All other organic solvents were used as received.
2.2. Phthaloylation of tryptophan The amino group of tryptophan was reacted with N-carbethoxyphthalimide according to [28,29]. Briey, to anhydrous sodium carbonate (0.52 g, 4.90 mmol) dissolved in 10 mL of water was added tryptophan (0.50 g, 2.45 mmol) under stirring. After complete dissolution, N-carbethoxyphthalimide (0.59 g, 2.69 mmol) was introduced into the reaction ask. The reaction mixture was further stirred for 2 h at room temperature and then extracted twice with ethyl acetate. The aqueous layer was acidied to pH 2e3 with 1 N hydrochloric acid solution and extracted three times with ethyl acetate. The combined organic layers were dried over anhydrous magnesium sulfate. The solvent was evaporated under reduced pressure after ltration. The residue is then puried

Table 1 Fed compositions of copolymerization of aCl3CL and 3CL. Copolymers Fed Weight (g) Fed Mole (mmol)

aCl3CL
P(3CL) P(10aCl3CL-903CL) P(20aCl3CL-803CL) P(30aCl3CL-703CL) e 0.29 0.59 0.89

3CL
2.28 2.05 1.82 1.60

aCl3CL
e 2 4 6

3CL
20 18 16 14

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13 C NMR (DMSO-d6): d 18.3 (CH2C^CH), 62.8 (OCH2C), 72.5 (C^CH), 80.6 (C^CH), 123.9 (CHCHCHCCO in pyridine), 125.5 (pyridine-CO), 136.8 (CHCHCHCCO in pyridine), 149.9 (NCHCCO in pyridine), 153.7 (CHNCHCCO in pyridine), 164.5 (C]O).

131.6 (2C, phthaloyl), 134.0 (2C, phthaloyl), 136.0 (indole), 167.5 (2C, phthaloyl), 168.9 (CHCOO). 2.4. Synthesis of a-chloro-3-caprolactone (aCl3CL) The aCl3CL monomer was synthesized according to the method of Lenoir et al. [30] with modication. Briey, to 12.5 g of mCPBA (50.7 mmol) dissolved in CH2Cl2 were added 5 g of a-chlorocyclohexanone (37.7 mmol) into the reaction ask. The reaction was conducted at room temperature for 48 h and then cooled down to 20  C to precipitate the white by-product which was removed by ltration. The ltrate was washed twice both with saturated sodium dithionite solution and with saturated sodium bicarbonate solution, and nally with water. Subsequently, the ltrate was dried over anhydrous magnesium sulfate, ltered, and concentrated under reduced pressure. Finally, the concentrated organic layer was puried by column chromatography on silica gel with the mobile phase Hex and EtOAc (1:3) yielding aCl3CL (3.9 g, 70%); TLC Rf 0.35; 1 H NMR (CDCl3): d 1.85 (m, 3H) 2.15 (m, 3H), 4.25 (m, 1H, COOCH2), 4.65 (m, 1H, COOCH2), 4.80 (dd, Jd 2.03 and 7.77 Hz, 1H, CHCl). 2.5. Synthesis of P(aCl3CL-ran-3CL) All glassware was dried in an oven overnight before use. The amounts of aCl3CL, and 3CL (see Table 1), 18 mg of 1,4-butanediol (0.2 mmol), and 81 mg of Sn(Oct)2 (0.2 mmol) were weighed into the reaction ask, which was evacuated for 15 min followed by purging with argon gas. Then the ask was immersed in an oil bath at 120  C. After 6 h of polymerization, the copolymer was recovered by precipitation in cold Hex and nally dried in vacuo for 24 h. In case of P(3CL) synthesis according 2.5, the homopolymerization of 3CL (2.28 g) was initiated with 18 mg of 1,4-butanediol and catalyzed by 81 mg of Sn(Oct)2. 2.6. Synthesis of P(aN33CL-ran-3CL) The azide substitution to P(aCl3CL-ran-3CL) was performed according to Riva et al. [12]. Briey, to 2 g of P(aCl3CL-ran-3CL) (1

2.3.2. Synthesis of but-3-ynyl 4-aminobenzoate (2) PABA (5 g, 36.5 mmol), 3.1 g (43.8 mmol) of 3-butyn-1-ol, 9.0 g (43.8 mmol) of DCC, and 5.3 g (43.8 mmol) of DMAP gave according to 2.3 an off-white solid. Puried compound 2 (4.5 g, 65%) was obtained through column chromatography on silica gel with the mobile phase of Hex and EtOAc (1:1) still as an off-white solid, m.p. 82.34  C, TLC with Rf 0.326, FTIR (KBr) n (cm1): 3398, 3336, 3288, 1685, 1599; 1 H NMR (CDCl3): d 2.04 (t, J 2.5 Hz, 1H, C^CH), 2.65 (td, J 6.8, 2.6 Hz, 2H, CH2C^CH), 4.09 (s, 2H, NH2), 4.38 (t, J 6.8 Hz, 2H, COCH2), 6.6 (d, J 8.5 Hz, 2H, benzene), 7.88 (d, J 8.5 Hz, 2H, benzene); 13 C NMR (CDCl3): d 19.1 (CH2C^CH), 62.0 (OCH2C), 69.8 (C^CH), 80.3 (C^CH), 113.7 (2C, benzene), 119.2 (benzene-CO), 131.6 (2C, benzene), 151.0 (benzene-NH2), 166.3 (C]O). 2.3.3. Synthesis of 3(2-N-phthalimido-2-(prop-2-ynyl acetayl) ethyl)indole (3) Phthaloyltryptophan (4.7 g, 14.0 mmol), 1.2 g (16.9 mmol) of 3butyn-1-ol, 4.4 g (21.1 mmol) of DCC, and 2.6 g (21.1 mmol) of DMAP gave according to 2.3 compound 3 (3.8 g, 70%) as a yellow solid after column chromatography on silica gel with the mobile phase of EtOAc, Hex and CH2Cl2 (1:2:3), m.p. 97.17  C, TLC with Rf 0.60, FTIR (KBr) n (cm1): 3411, 3270, 2120, 1777, 1738, 1714; 1 H NMR (CDCl3): d 1.85 (t, J 2.6 Hz, 1H, C^CH), 2.65 (m, 2H, CH2C^CH), 3.65 (d, J 8.0 Hz, 2H, CHCH2-indole), 4.70 (m, 2H, OCH2CH2), 5.30 (m, 1H, Phtaloyl-CHCH2-indole), 7.00 (s, 1H, CHNH in indole), 7.07 (td, J 7.04, 1.06 Hz, 1H, indole), 7.14 (td, J 8.07 1.1 Hz, 1 H, indole), 7.27 (dd, J 8.09, 0.68 Hz, 1H, indole), 7.62 (s, 1H, indole), 7.64 (m, 2H, phthaloyl), 7.75 (m, 2H, phthaloyl), 8.12 (s, 1H, NH); 13 C NMR (CDCl3): d 18.8 (CH2C^CH), 24.7 (CHCH2-indole), 52.6 (phthaloyl-CHCOO), 63.2 (COOCH2CH2), 70.0 (C^CH), 79.5 (C^CH), 110.8 (indole), 111.1 (indole), 118.4 (indole), 119.4 (indole), 122.0 (indole), 122.6 (indole), 123.3 (2C, phthaloyl), 127.1 (indole),

Fig. 3. General scheme for the synthesis of bioactive molecules grafted P(eCL). TEA as base was used for the synthesis of P((PABA-g-eCL)-ran-eCL.

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equiv of aCl3CL) dissolved in 5 mL DMF in a ask was added sodium azide (1.02 equiv). The reaction mixture was stirred overnight at room temperature. DMF was then completely evaporated under reduced pressure and the polymeric residue dissolved in toluene. The insoluble salt was centrifuged at 4000 rpm for 15 min. The clear supernatant was evaporated under reduced pressure resulting in the dry copolymer P(aN33CL-ran-3CL). 2.7. Typical click chemistry reaction Grafting of bioactive model compounds to P(3CL) backbone was carried out according to literature [12] with some modication. Briey, 200 mg of P(aN33CL-ran-3CL) (1 equiv of azide) was weighed into a reaction ask containing 5 mL of dry THF. Butynylester derivative of model compound (1, 2, or 3) (1.5 equiv), CuI (0.2 equiv), and base (0.2 equiv) were then loaded into the ask. Under atmospheric argon, the reaction was conducted at 40  C in a thermostatic bath for 4 h. The grafted copolymer was precipitated in cold Hex and dried in vacuo overnight before characterization. 2.8. Characterizations
1 H NMR spectra were recorded in CDCl3 or DMSO-d6 at 300 or 500 MHz, 13C NMR spectra in CDCl3 or DMSO-d6 at 75.45 MHz in the FT mode with Bruker Avance 300 apparatus at 25  C. Infrared spectra were recorded with Jasco FT/IR-4100 spectrophotometer. Number- and weight-average molecular weights (Mn and Mw, respectively) were measured by a Waters 150-CV gel-permeation chromatograph equipped with refractive index detector. THF was used as a mobile phase at a ow rate of 1 mL/min. Two columns of PLgel 10 mm mixed B were calibrated with polystyrene standards in the range of MW between 4490 and 1,112,000 g/mol. Thermal gravimetric analysis (TGA) was carried out with TGA 7 Perkin Elmer thermogravimetric analyzer at 10  C/min. Differential scanning calorimetry (DSC) was performed using DSC 7 Perkin Elmer

differential scanning calorimeter calibrated with indium. Glass transition and melting temperatures (Tg and Tm, respectively) were measured according to running condition: the sample was quenched to 80  C, heated to 100  C (rst heating), cooled down to 80  C, and heated again (second heating). Thermograms were recorded during the second heating cycle at 20  C/min. Mass spectrum was recorded in electron ionization mode at an ion source temperature of 150  C and an electron energy of 70 eV. Mass spectrum was scanned from 50 to 2000 m/z. 3. Results and discussion 3.1. Phthaloylation of tryptophan Tryptophan was phthaloylated prior to conversion to compound 3 through the reaction between tryptophan and N-carbethoxyphthalimide under basic condition in high product yield of 98%. 1H and 13C NMR data agreed with those of the literature [31,32]. The melting point was checked by DSC and the onset of Tm was found to be 172.83  C. CHN elemental analysis and mass spectrum agreed with the theoretical structure. In FTIR spectrum, the phthaloylation of amino group was characterized by disappearance of the strong primary NeH stretching peak of tryptophan at 3400 cm1; however, the weak secondary NeH stretching peak of indole was still observed. The two bands of C]O stretching at 1710 and 1774 cm1 conrmed the two carbonyls of phthaloyl group. The peak of C]O stretching of the carboxylic acid at 1710 cm1 was overlapped by that of the phthaloyl group. 3.2. Synthesis of esteried model compounds Typically, highly regioselective copper-mediated 1,3-dipolar cycloaddition between an azide group and a terminal alkyne requires an access of 1,2,3 triazoles [33]. Thus, in this study the feasible pathway was to x the azido group onto the polymer

Table 2 Molecular characteristic results of copolymers and grafted copolymers. Copolymers/Grafted copolymersa

aCl3CL or aN3 or ReChCH]/ [3CL] Molar Ratio in Feed

e 0.1 0.2 0.3 0.1 0.2 0.3 0.1 0.2 0.3 0.1 0.2 0.3 0.1 0.2 0.3

[aCl3CL or aN3 or ReChCH]/ [3CL] Calculated Molar Ratiob (Fgrafted) e 0.07 0.17 0.25 0.09 0.18 0.28 0.07 0.14 0.24 0.06 0.15 0.25 0.07 0.17 0.27

% Yield

Mn,theoc

Mn,NMRb

Mn,GPCd

Mw/Mnd

P(3CL) P(aCl3CL-ran-3CL) P(10aCl3CL-903CL) P(20aCl3CL-803CL) P(30aCl3CL-703CL) P(aN33CL-ran-3CL) P(10aN33CL-903CL) P(20aN33CL-803CL) P(30aN33CL-703CL) P((Nico-g-3CL)-ran-3CL) P((10Nico-g-3CL)-903CL) P((20Nico-g-3CL)-803CL) P((30Nico-g-3CL)-703CL) P((PABA-g-3CL)-ran-3CL) P((10PABA-g-3CL)-903CL) P((20PABA-g-3CL)-803CL) P((30PABA-g-3CL)-703CL) P((Phatryp-g-3CL)-ran-3CL) P((10Phatryp-g-3CL)-903CL) P((20Phatryp-g-3CL)-803CL) P((30Phatryp-g-3CL)-703CL)
a b

95.43 93.83 76.66 88.89 87.53 78.78 73.33 80.84 98.25 98.11 98.56 97.55 96.34 94.70 93.12 95.14

11,400 11,745 12,090 12,435 11,810 12,220 12,630 13,185 17,450 17,059 13,321 17,863 17,460 15,237 22,262 23,099

7737 8902 9085 6684 9258 10,226 8852 8034 8731 5155 12,533 13,098 11,052 19,789 21,708 24,788

9396 10,805 9927 7550 9869 9266 7133 11,246 7960 5675 10,455 19,663 18,047 13,982 15,858 12,029

1.90 1.96 2.24 2.20 2.09 2.29 2.27 1.70 1.83 1.62 1.88 1.53 1.75 1.56 1.87 1.88

Abbreviations: Nico nicotinic acid, PABA p-aminobenzoic acid, Phatryp phthaloyltryptophan. 1 Determined  by H NMR spectroscopy.  3 aCl3CLoraN3 3CLorAlkyne c Mn;theo CL Mn;aCl3CL or aN3 3CL or Alkyne This value was calculated based on 100% conversion of monomer; where [3CL] is the molar I 114 I concentration of 3CL, [aCl3CL or aN33CL or alkyne] is the molar concentration of aCl3CL and aN33CL and alkyne, respectively. [I] is the molar concentration of the initiator (1,4butanediol), and Mn;aCl3CL or aN3 3CL or alkyne is the molecular weight of aCl3CL and aN33CL and alkyne, respectively. d Determined by GPC.

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backbone (Fig. 3) and to esterify the model compound acids with 3butyn-1-ol using the coupling reaction with DCC and DMAP as coupling reagents (Fig. 2) to provide terminal alkynyl ester derivatives, which were characterized by NMR and FTIR spectroscopy as described below. 3.2.1. Synthesis of but-3-ynyl nicotinate (1) Under aforementioned conditions, but-3-ynyl nicotinate was obtained in 83% yield. Compound 1 was identied by 1H NMR using DMSO-d6 as solvent. The presence of the methyne proton signal of the terminal alkyne at 2.86 ppm and signal group of the methylene protons adjacent to the oxygen atom of ester group at 4.35 ppm in the spectrum conrmed the structure of but-3-ynyl nicotinate. This result was further veried by a FTIR spectrum. The CeH stretching peak of the terminal alkyne and C]O stretching band of ester carbonyl were identied at 3264 and 1718 cm1, respectively. Also a weak signal of the C^C stretching was observed at 2119 cm1. 3.2.2. Synthesis of but-3-ynyl 4-aminobenzoate (2) The coupling reaction according to 3.2 between PABA and 3butyn-1-ol gave but-3-ynyl 4-aminobenzoate in 65% yield. Compound 2 was identied by 1H NMR in CDCl3. The spectrum showed again the typical signal groups for the methylene protons (COOCH2) at 4.38 ppm and the methyne proton (C^CH) at 2.04 ppm. The FTIR spectrum recorded in the KBr disc mode showed also the CeH stretching of the terminal alkyne at 3288 cm1 and, in addition, the NeH stretching of the anilino group around 3400 cm1. The peaks at 1685 and 1599 cm1 were assigned to the C]O stretching and NeH bending, respectively. 3.2.3. Synthesis of 3(2-N-phthalimido-2-(prop-2-ynyl acetayl) ethyl)indole (3) Phthaloyltryptophan (see 3.1) was esteried with 3-butyn-1-ol to provide the terminal alkyne functional group according to 3.2 in 70% yield. Compound 3 was identied by 1H NMR in CDCl3. The spectrum exhibited the characteristic signal group for the methylene protons (COOCH2) at 4.70 ppm of the ester linkage and that of the alkyne proton at 1.85 ppm. The FTIR spectrum showed the corresponding CeH stretching of the C^CH group at 3270 cm1 and the C]O stretching peak at 1738 cm1.

3.4. Synthesis of P(aN33CL-ran-3CL) Conversion of the chloride pendants to azides was done prior to grafting the model compounds along the polymer chain. P(aCl3CLran-3CL) was substituted with sodium azide in DMF at room temperature (Fig. 3) in an overnight reaction. The 1H NMR spectrum conrmed the complete conversion by means of the azide methine signal (CHN3) at 3.85 ppm and the entire disappearance of methyne proton signal (CHCl) at 4.25 ppm. The azide group was also monitored by FTIR by means of the characteristic band at 2100 cm1. The molecular weight of P(aN33CL-ran-3CL) was slightly increased due to the azide substitution compared to that of P (aCl3CL-ran-3CL) (Table 2).

3.3. Synthesis of P(aCl3CL-ran-3CL) The aCl3CL unit was copolymerized at various molar ratios (0.10, 0.20, and 0.30) with 3CL monomer by ring opening polymerization in the presence of the initiator 1,4-butanediol and the catalyst Sn (Oct)2 (Fig. 3). The molar ratio of monomer to initiator and to catalyst, respectively, was set to 100 each. The yield of the copolymers was in each case higher than 70% with molar ratios of the aCl3CL units calculated from 1H NMR which were in agreement with the theoretical values. The Mn,theo values were calculated based on 100% conversion of the monomer, the Mn,NMR values in Table 2 according to [30] with some modication. Both sets differ to a large extent from each other. This fact might be explained by the electron withdrawing property of the chloride atom in aCl3CL leading to a higher reactivity of its lactone moiety in comparison to that of 3CL. This caused additional inter- and intramolecular transesterications during the optimized polymerization process at the high temperature of 120  C, which were particularly inevitable at the high 0.3 ratio of aCl3CL versus 3CL. The complete molecular parameter set of the copolymers and grafted copolymers are compiled in Table 2.

Fig. 4. FTIR spectra of P((Nico-g-3CL)-ran-3CL) (A), P((PABA-g-3CL)-ran-3CL) (B), and P ((Phatryp-g-3CL)-ran-3CL) (C), respectively, in comparison with P(3CL) homopolymer, P (aCl3CL-ran-3CL), P(aN33CL-ran-3CL), and alkyne terminated drug molecules. DBU as base was used for the syntheses of (A) and (C), respectively, whereas TEA for that of (B).

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Fig. 5. 1H NMR spectra of P((Nico-g-3CL)-ran-3CL) (A), P((PABA-g-3CL)- ran-3CL) (B), and P((Phatryp-g-3CL)-ran-3CL) (C), respectively. DBU as base was used for the syntheses of (A) and (C), respectively, whereas TEA for that of (B).

3.5. Engrafting of model compounds along P(aN33CL-ran-3CL) by click reaction The 3-butynyl ester derivatives of the three model compounds nicotinic acid, PABA, and phthaloyltryptophan were engrafted onto

P(aN33CL-ran-3CL) backbone through copper-catalyzed Huisgens 1,3-dipolar cycloaddition reaction at 40  C using CuI as catalyst and DBU as base, (Fig. 3). Under monitoring by FTIR, the reaction was completed after 4 h with the complete disappearance of azide peak at 2100 cm1 indicating the entire conversion of the pendent azide

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to the triazole ring which showed the characteristic peak at 1610 cm1. Fig. 4 illustrates the FTIR spectra of all grafted copolymers. In addition, the 1H NMR spectrum conrmed the cycloaddition by the methyne proton signal of the triazole ring at 7.7 ppm and the disappearance of the methyne proton signal of azide (CHN3) at 3.85 ppm. The average molecular weight (Mn,NMR) of the grafted copolymers and the molar fraction (Fgrafted) of the engrafted model compounds were calculated according to equation (1) and 2, where I represents the integration of the respective 1H NMR peaks assigned in Fig. 5, and M the molecular weight of compound 1, 2, 3 (Malkyne), and the caprolactone unit (M3CL), respectively. All values are summarized in Table 2 together with the calculated grafting efciencies (Fgrafted) which were higher than 80%. Equation (1) For nicotinic acid and PABA grafted P(3CL)

Mn;NMR

h i h i IA Malkyne 1 I M 3 D CL 2
1I 4 H

For phthaloyltryptophan grafted P(3CL)

Fig. 6. GPC chromatograms of P((PABA-g-eCL)-co-eCL) at 30% mole of PABA representing the products of the cycloaddition with TEA and DBU (d,),respectively, as base.

Mn;NMR

0h 1 i IAL Malkyne ID M3CL B C 2@ A IHM IAL

Equation (2) For nicotinic acid and PABA grafted P(3CL)

FgraftedCL

1I 2 D

IA IA

For phthaloyltryptophan grafted P(3CL)

FgraftedCL

IAL ID IAL

3.5.1. Grafting of but-3-ynyl nicotinate (1) along the P(3CL) backbone But-3-ynyl nicotinate (1) was successfully grafted onto the polymer backbone. Fig. 5A shows the 1H NMR spectrum of nicotinic acid grafted P(3CL) with the characteristic peak of methyne proton of caproyl unit adjacent to the triazole ring at 5.30 ppm (peak A), while the former signal of the methyne proton adjacent to the azide at 3.85 ppm was missing. Further new peaks at 7.45, 8.30, 8.70 and 9.25 ppm were assigned to the protons of nicotinic pyridine ring. These results were conrmed by characteristic peaks of C]C stretching at 1590 cm1, and of C]N stretching at 1625 cm1 in the FTIR spectrum originating from the overlapping vibrations of the C]N bonds of the triazole and pyridine rings (Fig. 4A). The molecular weights of the grafted polymers declined only to a minor extent as compared to those of P(aN33CL-ran-3CL) and P (aCl3CL-ran-3CL) (Table 2). In case of the grafted nicotinic acid, at low percent grafting, the Mn value was remarkably lower for 30% grafting. However, the molecular weight distribution was narrower than those of the original polymers and the value of Mw/Mn and GPC chromatogram showed a unimodal peak (data not shown). If one considers the difference of Mn,theo and Mn,NMR, degradation must have occurred. The nitrogen atom of the nicotinic pyridine ring acts as an intramolecular nucleophile with its localized lone pair electron, cleaving the ester bond by attacking the carbonyl group during the grafting step independent of the strength of the base used. Therefore the extent of polymer degradation could not be minimized, yielding short polymer units which could not be precipitated by cold hexane. This, nally, explains the unimodal GPC peak.

3.5.2. Grafting of but-3-ynyl 4-aminobenzoate (2) along the P(3CL) backbone For grafting but-3-ynyl 4-aminobenzoate (2) onto the polymer backbone, DBU was initially used and acted as a base in the cycloaddition reaction. Similar to grafting of nicotinic acid, the reaction was complete after 4 h. At 10% grafting, the corresponding grafted copolymer was attainable without degradation as seen by an increase in Mn,GPC without broadening the polydispersity index of molecular weight (PDI) compared to that of P(10aN33CL-ran3CL). Once the percent grafting of but-3-ynyl 4-aminobenzoate (2) was increased to 20 and 30% in the presence of the strong nonnucleophilic, sterically hindered base DBU [34,35], the PDI grew dependently, only slightly raised for the 20% grafting copolymer (data not shown). In contrast, for 30% grafting the GPC chromatogram showed a bimodal peak (Fig. 6), indicating a heterodispersed molecular weight arising from polymer degradation by DBU base
Table 3 Thermal parameters of copolymers and grafted copolymers determined by DSC thermograms. Copolymers/Grafted copolymersa P(3CL) P(aCl3CL-ran-3CL) P(10aCl3CL-903CL) P(20aCl3CL-803CL) P(30aCl3CL-703CL) P(aN33CL-ran-3CL) P(10aN33CL-903CL) P(20aN33CL-803CL) P(30aN33CL-703CL) P((Nico-g-3CL)-ran-3CL) P((10Nico-g-3CL)-903CL) P((20Nico-g-3CL)-803CL) P((30Nico-g-3CL)-703CL) P((PABA-g-3CL)-ran-3CL) P((10PABA-g-3CL)-903CL) P((20PABA-g-3CL)-803CL) P((30PABA-g-3CL)-703CL) P((Phatryp-g-3CL)-ran-3CL) P((10Phatryp-g-3CL)-903CL) P((20Phatryp-g-3CL)-803CL) P((30Phatryp-g-3CL)-703CL) Tg ( C) Ndb 74.7 52.6 55.9 58.1 46.8 49.9 30.1 27.5 13.6 41.0 13.2 5.6 15.1 e e Tm ( C) 50.9 35.2 36.4 12.1 37.8 17.6 19.7 38.6 ec e 37.1 e e e e e acid,

Tg and Tm stand for glass transition and melting temperatures, respectively. a Abbreviations: Nico nicotinic acid, PABA p-aminobenzoic Phatryp phthaloyltryptophan. b Nd Not determined. c e no peak observed.

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catalyzed nucleophilic ester cleavage in the polymer backbone caused by the deprotonated amino group of the PABA aniline fragment. This is leading to intra- and intermolecular transesterications. When the milder base triethylamine (TEA) was used instead of DBU, the extent of degradation of the easily degraded polymer backbone was diminished. Therefore, TEA was further on used in this study, leading to successful grafting PABA onto the polymer

chain. Under this modied condition the grafted copolymers at 20 and 30% showed molecular weights which increased with the same molecular weight distribution as compared to P(aN33CL-ran-3CL) (Table 2). The GPC chromatogram conrmed this result with a unimodal in stead of the bimodal peak. The grafted copolymer was characterized by the 1H NMR signal of the methyne proton of the triazole ring at 7.65 ppm and those of the benzene protons of PABA at 6.65 and 7.85 ppm (Fig. 5B). The

Fig. 7. TGA proles of copolymers and grafted copolymers at 20% molar ratio (d %weight loss; 1st derivative).

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grafting was also conrmed by the FTIR spectrum with bands for NeH stretching at 3400 cm1, C]N stretching of the triazole ring at 1626 cm1, and C]O stretching of the polyester polymer and ester bond of the pendent PABA, at 1700 and 1725 cm1, respectively (Fig. 4B). 3.5.3. Grafting of phthaloyltryptophan by means of 3(2-Nphthalimido-2-(prop-2-ynyl acetayl)ethyl)indole (3) along the P(3CL) backbone The phthaloyltryptophan by means of 3(2-N-phthalimido-2(prop-2-ynyl acetayl)ethyl)indole (3) was used as a large and bulky representative to investigate the limitation of grafting onto P(3CL) backbone through click reaction. However, this compound could be completely attached along P(3CL) after 4 h using the same conditions as for engrafting of nicotinic acid. The 1H NMR spectrum showed the characteristic signal of triazole proton (Fig. 5C), together with the typical set of signal groups of phthaloyltryptophan between 6.9 and 8.0 ppm coinciding with the absence of methyne proton signal of the former azide (CHeN3) at 3.85 ppm. The FTIR spectrum (Fig. 4C) also conrmed the successful click reaction by means of the NeH stretching peak at 3400 cm1 and the two C]O stretching bands at 1710 and 1774 cm1 of the phthaloyl group. The molecular weight of the grafted copolymer calculated from 1 H NMR and GPC gradually increased according to the increasing number of grafted units. Its distribution was narrower than that of P (aN33CL-ran-3CL) (Table 2). Unlike to the grafted PABA, DBU did not cause any severe chain degradation according to GPC results, since the indole ring proton is less acidic and sterically hindered by the phenyl ring and hardly converted to the nucleophilic species. However, this steric effect did not disturb the completeness of grafting reaction. In contrast, Riva et al. [12] were only able to engraft a linear macromolecule, PEO, on the P(3CL) backbone to just 40% probably due to stronger sterical interaction between two macromolecules than in the case of phthaloyltryptophan with complete of engrafting. 3.6. Thermal properties of the copolymers and grafted copolymers The thermal properties of the grafted copolymers were measured with DSC and TGA and compared to those of the original P(aN33CL-ran-3CL) and P(aCl3CL-ran-3CL). The glass transition temperature Tg and melting temperature Tm values characterizing the polymorphic properties of the copolymers are summarized in Table 3 indicating their semisolid form. In case of an undetectable Tms the polymer is amorphous. In some cases of destructible polymers Tgs cannot be detected. The Tgs values of P(aN33CL-ran3CL) and of P(aCl3CL-ran-3CL) tend to increase with increasing molar ratio of substituted units, meanwhile their Tms tend to decline. The increase of grafted units in the copolymer resulted in the change of the polymorphic form from semi-crystalline to amorphous, altering their thermal properties. At 10% grafting of nicotinic acid and PABA, respectively, the Tm was observable while at higher ratios it was undetectable. In case of grafting phthaloyltryptophan, Tm was not detected at all grafting ratios. Thus, conversion from polymorphic to amorphous form of these kinds of copolymers goes with their size and/or with the increasing number of substituted units. To investigate the thermal degradation of the copolymers in comparison with the grafted copolymers, TGA proles were recorded. Fig. 7 illustrates TGA proles of copolymers and grafted copolymers at 20% molar ratio. The thermograms of P(aN33CL-ran-3CL) and P(aCl3CL-ran-3CL) show different thermal degradation pattern. Although, the starting degrading temperature of both copolymers at around 200  C was similar, in case of P(aCl3CL-ran-3CL) the main

degradation peak appeared at around 335  C followed by a small degradation peak, a moderate degradation peak at 370  C, and by a small broad degradation peak starting at 460  C. In contrast, P (aN33CL-ran-3CL) showed a small degradation peak at 270  C attributed to the initial rupture of azide (NeN2) [36,37] and an extensive weight loss was found at 350  C followed by moderate degradation peaks at 400  C. In case of the grafted copolymers, the thermal degradation pattern of P((Nico-g-3CL)-ran-3CL was the same as that of P(aN33CL-ran-3CL). The thermogram of P((PABA-g-3CL)ran-3CL exhibited only two main degradation peaks, an extensive at 320  C and a moderate one at 370  C. the latter one being stronger than that of P(aN33CL-ran-3CL) at comparable temperature. For P ((Phatryp-g-3CL)-ran-3CL, the extensive degradation started at 370  C while the second degradation step was shifted to about 550  C. According to these results all copolymers and grafted copolymers are thermally stable to about 200  C in dried state. From these combined results it can be concluded that the thermal properties of the grafted copolymers are largely governed by the type of grafted bioactive model compound at the polymer backbone. The larger bioactive molecule altered the thermal properties stronger than the smaller type. In addition, an increasing amount of grafted model compound changed the structure of the grafted copolymer to the amorphous form. 4. Conclusion Three bioactive model compounds (nicotinic acid, PABA, and phthaloyltryptophan) were successively grafted onto the P(3CL) backbone through copper-catalyzed Huisgens 1,3-dipolar cycloaddition or the click reaction. Under very mild conditions, the reaction could preserve the length of the polymer backbone. However, reecting the type of the model compound containing either a protic or a nucleophilic group the type of base used in the reaction is the critical issue since in combination they may possibly lead to intra- or intermolecular transesterication and thus induce the polymer chain degradation. Several types of model compounds could be grafted along the polymer backbone by varying amounts of the aCl3CL units on the backbone. The molecular size of the compounds modied the physicochemical and thermal properties of the grafted copolymers compared to the copolymer. The grafted copolymers could be identied by FTIR, NMR, and GPC analysis. Derived from this accomplishment, various kinds of drugs including cytotoxic drugs, peptides and proteins, could probably be grafted along the polymer backbone in ongoing research in order to modify the physicochemical properties of drugs and also of macromolecular carriers to be more suitable for drug delivery applications. The conjugation of these grafted copolymers with other polymeric carriers such as PEG and nanoparticles prepared from these grafted copolymers are being under investigation to gain more efcient drug delivery systems. Acknowledgements Financial support from the Thailand Research Fund (TRF) through the Royal Golden Jubilee Ph.D. Program (Grant No. PHD/ 0189/2547) and the Thai Research Fund and Commission of Higher Education, Thailand for the research funding (RMU5180019) is gratefully acknowledged. The authors are very pleased to acknowledge the National Metal and Materials Technology Center (MTEC, Pathumthani, Thailand) for GPC experiment. The authors thank Professor Dr. AW Frahm, Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmaceutical Sciences, Freiburg University, Freiburg, Germany, for language and writing approval.

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Appendix. Supplementary material Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.polymer.2010.03.034. References
[1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] Lee JS, Go DH, Bae JW, Lee SJ, Park KD. J Control Release 2007;117(2):204e9. Park EK, Kim SY, Lee SB, Lee YM. J Control Release 2005;109(1e3):158e68. Jubo L, Payam Z, Faquan Z, Christine A. J Pharm Sci 2008;97(8):3274e90. Shi Q, Chen X, Lu T, Jing X. Biomaterials 2008;29(8):1118e26. Kulbokaite R, Ciuta G, Netopilik M, Makuska R. Reactive Funct Polym 2009;69 (10):771e8. Brner HG. Prog Polym Sci 2009;34(9):811e51. Trollsas M, Lee VY, Mecerreyes D, Lowenhielm P, Moller M, Miller RD, et al. Macromolecules 2000;33(13):4619e27. Rieger J, Bustele KV, Lecomte P, Detrembleur C, Jrme R, Jrme C. Chem Commun (Camb) 2005;2:274e6. Lecomte P, Riva R, Schmeits S, Rieger J, Butsele KV, Jrme C, et al. Macromol Symp 2006;240:157e65. Pitt CG, Gu Z-W, Ingram P, Hendren RW. J Polym Sci A Polym Chem 1987;25 (4):955e66. Lutz J-F, Zarafshani Z. Adv Drug Deliv Rev 2008;60(9):958e70. Riva R, Schmeits S, Jrme C, Jrme R, Lecomte P. Macromolecules 2007;40 (4):796e803. Riva R, Lussis P, Lenoir S, Jrme C, Jrme R, Lecomte P. Polymer 2008;49 (8):2023e8. Zednik J, Riva R, Lussis P, Jrme C, Jrme R, Lecomte P. Polymer 2008;49 (3):697e702. Lee R-S, Huang Y- T. J Polym Sci A Polym Chem 2008;46(13):4320e31.

[16] Chen A-L, Ni H-C, Wang L-F, Chen J- S. Biomacromolecules 2008;9 (9):2447e57. [17] Pertuit D, Moulari B, Betz T, Nadaradjane A, Neumann D, Ismali L, et al. J Control Release 2007;123(3):211e8. [18] Bingham S, Cummings JH. Clin Sci (Lond) 1983;64(6):629e35. [19] Brueckner B, Boy RG, Siedlecki P, Musch T, Kliem HC, Zielenkiewicz P, et al. Cancer Res 2005;65(14):6305e11. [20] Guo B, Zhou X, Xiao X, Li J, Li L. Hua Xi Kou Qiang Yi Xue Za Zhi 2001;19 (5):315e7. [21] Iinuma I, Uyama S, Uenoyama K, Sakaguchi T. Nippon Ganka Gakkai Zasshi 1961;65:1463e7. [22] Jakobsen J, Pedersen AN, Ovesen L. Eur J Clin Nutr 2003;57(1):138e42. [23] Pahan K. Cell Mol Life Sci 2006;63(10):1165e78. [24] Safeer RS, Lacivita CL. Am Fam Physician 2000;61(11):3371e82. [25] Siedlecki P, Boy RG, Musch T, Brueckner B, Suhai S, Lyko F, et al. J Med Chem 2006;49(2):678e83. [26] p-Aminobenzoic acid. In: Windholz M, Budavari S, editors. The Merck index. 10th ed. New Jersey: Merck & Co., Inc.; 1983. p. 62e3. [27] Kern CJ, Antoshkiw T, Maiese MR. Anal Chem 2002;20(10):919e22. [28] Nefkens GHL. Nature 1960;185(4709):309. [29] Nefkens GHL, Tesser GI, Nivard RJF. Recl Trav Chim Pays Bas 1960;79: 688e98. [30] Lenoir S, Riva R, Lou X, Detrembleur C, Jrme R, Lecomte P. Macromolecules 2004;37(11):4055e61. [31] Mizrahi DM, Waner T, Segall Y. Phosphorus, Sulfur Silicon Relat Elem 2001;173(1):1e25. [32] Zeng Q, Liu Z, Li B, Wang F. Amino Acids 2004;27(2):183e6. [33] Gil MV, Arevalo MJ, Lopez O. Synthesis 2007;2007(11):1589e620. [34] Im YJ, Gong iH, Kim HJ, Kim JN. Bull Korean Chem Soc 2001;22(9):1053e5. [35] Oediger H, Mller F, Eiter K. Synthesis 1972;1972(11):591e7. [36] Ero MS, Gven O. J Appl Polym Sci 1996;61(2):201e6. [37] FazlIoglu H, Hacaloglu J. J Anal Appl Pyrol 2002;63(2):327e38.